Alloantigen presenting function of normal human CD34+ hematopoietic cells

The identification of the CD34 molecule, expressed almost exclusively on human hematopoietic stem cells and committed progenitors, and the development of CD34-specific monoclonal antibodies have made procurement of relatively pure populations of CD34+ marrow cells for autologous transplantation feasible. Characterization of the immunogenicity of CD34+ marrow cells may facilitate the design of successful strategies to use these cells for allogeneic transplantation. CD34+ marrow cells from normal volunteers were enriched to greater than 98% purity by immunoaffinity chromatography on column followed by fluorescence-activated cell sorting. Purified CD34+ cells were tested for expression of HLA-DR and other accessory molecules, and function in hematopoietic colony growth and mixed leukocyte culture (MLC) assays. Greater than 95% CD34+ cells were positive for HLA-DR and 74% +/- 10% were highly positive for CD18, the common beta-chain of a leukointegrin family. CD34+/CD18- cells were small, agranular lymphocytes which contained the majority of precursors for colony-forming cells detected in long-term cultures. They produced almost no stimulation of purified T cells from HLA-DR-incompatible individuals in bulk MLC or in limiting dilution assay. In contrast, CD34+/CD18+ cells were large, were enriched for cells forming mixed colonies in short- but not long-term assays, and were capable of stimulating allogeneic T cells. CD86, a natural ligand for the T-cell activation molecule CD28, was coexpressed with CD18 in 6% +/- 3% of CD34+ cells. CD34+/CD86+ cells, but not CD34+/CD86- cells, exhibited strong alloantigen presenting function. Thus, pluripotent hematopoietic activity and alloantigen presenting function are attributes of distinct subsets of CD34+ marrow cells. CD34+/CD18- or CD34+/CD86- cells may be more effective than either the whole CD34+ population or unseparated marrow in engrafting allogeneic recipients and may also facilitate induction of tolerance.

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Evaluation of the Phenotype and GVHD-Inducing Potential of Splenic T Cells Isolated from G-CSF, AMD3100, or G-CSF and AMD3100 Pretreated Allogeneic Donors.

Blood ◽

10.1182/blood.v106.11.5224.5224 ◽

2005 ◽

Vol 106 (11) ◽

pp. 5224-5224

Author(s):

Michael Rettig ◽

Matthew Holt ◽

Fulu Liu ◽

Julie Ritchey ◽

Daniel Link ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Peripheral Blood ◽

Cell Activation ◽

Cell Function ◽

Single Injection ◽

Lower Percentage ◽

Hematopoietic Stem ◽

Cd34 Cells

Abstract Recent data suggest that the incidence and severity of acute graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell (HSC) transplantation may depend on the agent used to mobilize HSC from the bone marrow (BM) to the peripheral blood. AMD3100 is a bicyclam that selectively and reversibly blocks CXCL12 (SDF-1) binding to, and signaling through, CXCR4. We and others have previously demonstrated that AMD3100 induces the rapid mobilization of both murine and human HSCs, causing up to a 10-fold increase in CD34+ cells in the peripheral blood within 4–6 hours of a single injection. Furthermore, a single dose of AMD3100 can be combined with a well-tolerated dose of G-CSF on the fifth day of HSC mobilization to markedly increase the mobilization of CD34+ cells. Since T cells from donors treated with G-CSF reportedly have a reduced capacity to induce GVHD relative to those from control-treated donors, we analyzed the phenotype and GVHD potential of T cells isolated from the spleens of donors that were unmobilized or treated with G-CSF, AMD3100, or G-CSF and AMD3100. C57BL/6 mice were treated with G-CSF (250 μg/kg/d for 5 days), AMD3100 (single injection of 5 mg/kg), or G-CSF + AMD3100 (AMD3100 only given on the final day of G-CSF). Following mobilization, we purified splenic T-lymphocytes to >95% by negative immunoselection and examined expression of CD4, CD8, CD25, CD69, CD30, α4β7, CD44, and CD62L on CD3+ cells by flow cytometry. Although mobilization did not significantly alter the percentage of CD4+, CD8+ or CD4+CD8+ T cells in the spleen, there was a trend toward a lower percentage of splenic CD4+CD25+CD62L+ naturally occurring T regulatory cells following G-CSF and/or AMD3100 administration. Similar to previous reports by others, we observed that the percentage of CD4+ and CD8+ cells that expressed CD62L and the level of CD62L expression was significantly decreased in G-CSF-treated donors (both with and without AMD3100) compared with the untreated control. This decrease in the expression of CD62L was not observed on T cells isolated from donors that were treated with AMD3100 alone. Furthermore, the reduction in CD62L expression on T cells isolated from G-CSF-treated donors did not coincide with an increase in the expression of CD44, suggesting that the loss of CD62L was not due to an expansion of memory T cells. In fact, all three mobilization regimens induced a significant decrease in the percentage of CD44hi expressing splenic T cells. Consistent with this observation, G-CSF and/or AMD3100 administration did not induce T cell activation as assessed by CD25, CD30 and CD69 expression. Similarly, we found no alterations in expression of the intestinal homing receptor, α4β7, following donor mobilization with G-CSF and/or AMD3100. To evaluate the GVHD-inducing potential of the purified T cells, we injected 5e5 or 2e6 total T cells, along with T cell depleted C57BL/6 BM, into lethally irradiated BALB/c recipients. Surprisingly, GVHD-related weight loss, median survivals and lymphoid reconstitution were identical in each group using Kaplan-Meier and Mantel/Cox log rank analyses. These data clearly suggest that AMD3100 alone or when combined with G-CSF as a mobilizing agent has no significant effect on T cell function as measured by GVHD studies. These studies further support the use of AMD3100 for the mobilization of allogeneic stem cells.

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T Cell Activation By OKT3 Is a Function of the Percent Monocytes in PBMC of Healthy Donors and MDS Patients

Blood ◽

10.1182/blood-2019-131474 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4870-4870

Author(s):

Alison Tarke ◽

Valentina Ferrari ◽

Hannah Fields ◽

Luca Ferrari ◽

Franco Ferrari ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Cell Transplant ◽

Activation Markers ◽

Hematopoietic Stem ◽

Hla Dr ◽

Healthy Donors

Background: Myelodysplastic Syndromes (MDS) are a heterogeneous hematologic malignancy characterized by bone marrow failure and cytopenias. The median survival rate for patients with higher-risk MDS who fail standard-of-care chemotherapy with hypomethylating agents (HMAs) is less than 6 months, and the only curative treatment for these patients is hematopoietic stem cell transplant (HSCT). Over the past 10 years, immunotherapy as a cancer treatment has achieved variable levels of success in different tumor types. There are currently 22 active clinical trials of immunotherapies for MDS (www.clinicaltrials.gov; 7/30/19), including our phase I clinical trial with a personalized adoptive cellular therapy targeting MDS patient neoantigens (NCT 03258359). Because MDS patients are frequently monocytopenic and the existing literature is inconsistent regarding the ability of MDS patients' monocytes to support T cell activation, we compared the activation of MDS T cells with those of healthy donors in the presence of autologous monocytes. Methods: Peripheral blood mononuclear cells (PBMC) from 5 healthy donors and 7 higher-risk MDS patients were cryopreserved after Ficoll separation. These PBMC were thawed and aliquoted into 6 replicate wells of 200,000 cells in 96-well u-bottom plates in R-10 culture medium. Half of the wells were treated with 25 ng/mL OKT3 and 200 U/mL IL-2. After 48 hours at 37˚C with 5% CO2, the wells were collected for analysis by flow cytometry. Beads were used to detect T cell activation induced secretion of IFNg, TNFa, IL-4, IL-10, and IL-17 in the supernatant and fluorescent antibodies were used to phenotype viable cells for CD3, CD4, CD8, and the T cell activation markers, CD69, CD25, CTLA-4, PD-1, and HLA-DR. Results: We measured a higher release of IFNg and TNFa in donor PBMC compared to MDS patients after OKT3/IL-2 activation, p < 0.01 and 0.04, respectively by 2-way ANOVA. The expression of CD69, CD25, HLA-DR, and CTLA4 increased variably on activated T cells from donors or MDS patients, but expression of CD4+CD25+ was more frequent on donor T cells after activation (p = 0.03). Activation also resulted in a higher frequency of PD-1 expression on donor CD4+ and CD8+ T cells than on MDS T cells (p < 0.01 and < 0.01, respectively). Interestingly, on both MDS and normal T cells the percentage of CD8+PD1+ activated cells correlated strongly with the percent of CD14+ monocytes present in the PBMC (R2 = 0.92 and 0.60 respectively; Fig 1a and 1b). We designed further experiments to test whether this was a patient intrinsic phenomenon, or if the absolute number of CD14+ monocytes in the PBMC was associated with different levels of PD1 expression upon T cell activation. First, we separated CD14+ cells from the PBMC of a patient with MDS using magnetic beads. Then CD14+ cells were added back to the CD14-depleted PBMC at a final percent of 0.5, 5, 10, 20, 35, 70, or 100% of the original amount. Unmodified PBMC was included as a control and all cells were stimulated with OKT3 and IL-2 or left in R-10 medium without stimulus. After 24, 48, and 70 hours, samples were collected to analyze by flow cytometry for CD3, CD4, CD8, CD14, and PD1 expression. The results show that an increasing percent of monocytes corresponded to the increased expression of PD1 on CD8+ and CD4+ T cells. Conclusion: Our results show that there are variable reductions in markers of T cell activation and cytokine secretion in MDS patients compared to healthy donors. We also observed that the fold increase in activation induced PD-1 expression was well correlated with the percent of CD14+ monocytes in the PBMC of both MDS patients and healthy donors. Direct experimentation revealed that this correlation is a cause-effect relationship. We are continuing to investigate the role of monocytes in T cell activation in MDS patients. Disclosures Bejar: Celgene: Consultancy; Takeda Pharmaceuticals: Research Funding; AbbVie/Genentech: Consultancy, Honoraria; Astex/Otsuka: Consultancy; Modus Outcomes: Consultancy; Daiichi-Sankyo: Consultancy. Lane:PersImmune, Inc.: Employment.

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HIV-specific T-cell Responses and Generalized Activation in HIV-1 Infected Long-term Non-progressors and Progressors from South India

Current HIV Research ◽

10.2174/1570162x17666181212122607 ◽

2019 ◽

Vol 16 (4) ◽

pp. 302-314

Author(s):

Chinnambedu Ravichandran Swathirajan ◽

Ramachandran Vignesh ◽

Greer Waldrop ◽

Uma Shanmugasundaram ◽

Pannerselvam Nandagopal ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Cd4 T Cell ◽

Cell Responses ◽

Activation Rates ◽

Hiv 1

Background:Anti-viral cytokine expressions by cytotoxic T-cells and lower activation rates have been reported to correlate with suppressed HIV replication in long-term non-progressors (LTNP). Immune mechanisms underlying disease non-progression in LTNP might vary with HIV-1 subtype and geographical locations.Objective:This study evaluates cytokine expression and T-cells activation in relation to disease non-progression in LTNP.Methods:HIV-1 Subtype C infected LTNP (n=20) and progressors (n=15) were enrolled and flowcytometry assays were performed to study HIV-specific CD8 T-cells expressing IL-2, IFN-γ, TNF-α and MIP-1β against gag and env peptides. CD4+ T-cell activation was evaluated by surface expression of HLADR and CD38.Results:Proportions of cytokines studied did not differ significantly between LTNP and progressors, while contrasting correlations with disease progression markers were observed in LTNP. CD4+ T-cell activation rates were significantly lower in LTNP compared to progressors which indicate the potential role of T-cell activation rates in disease non-progression in LTNP.Conclusion:LTNP and progressors showed similar CD8+ T-cell responses, but final conclusions can be drawn only by comparing multiple immune factors in larger LTNP cohort with HIV-1 infected individuals at various levels of disease progression. A possible role of HIV-1 subtype variation and ethnic differences in addition to host-genetic and viral factors cannot be ruled out.

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Autologous transplantation of canine long-term marrow culture cells genetically marked by retroviral vectors

Blood ◽

10.1182/blood.v79.2.356.356 ◽

1992 ◽

Vol 79 (2) ◽

pp. 356-364 ◽

Cited By ~ 89

Author(s):

RF Carter ◽

AC Abrams-Ogg ◽

JE Dick ◽

SA Kruth ◽

VE Valli ◽

...

Keyword(s):

Gene Transfer ◽

Autologous Transplantation ◽

Large Animal Model ◽

Multiple Infections ◽

Large Animal ◽

Retroviral Infection ◽

Hematopoietic Stem ◽

Marrow Cells

Abstract Retroviral infection of bone marrow cells in long-term marrow cultures (LTMCs) offers several theoretical advantages over other methods for gene transfer into hematopoietic stem cells. To investigate the feasibility of this approach in a large animal model system, we subjected LTMCs from nine dogs to multiple infections with retrovirus containing the neomycin phosphotransferase gene (neo) during 21 days of culture. Feeder layers, cocultivation, polycations, and selection were not used. The in vitro gene transfer efficiency was 70% as determined by polymerase chain reaction amplification of neo sequences in colony- forming unit granulocyte-macrophage (CFU-GM) obtained from day-21 LTMCs. Day-21 LTMC cells were infused into autologous recipients with (four dogs) and without (three dogs) marrow-ablative conditioning. At 3 months posttransplant, up to 10% of marrow cells contained the neo gene. This percentage declined to 0.1% to 1% at 10 to 21 months posttransplant. Neo was also detected in individual CFU-GM, burst- forming unit-erythroid (BFU-E), and CFU-Mix progenitors derived from marrow up to 21 months postinfusion and in cultures of peripheral blood- derived T cells up to 19 months postinfusion. There was no difference in the percentage of neo-marked cells present when dogs that received marrow ablative conditioning were compared with dogs receiving no conditioning. Detection of neo-marked marrow cells almost 2 years after autologous transplantation in a large mammalian species shows that retroviral infection of marrow cells in LTMCs is a potentially nontoxic and efficient protocol for gene transfer. Further, our results suggest that marrow conditioning and in vivo selection pressure to retain transplanted cells may not be absolute requirements for the retention of genetically marked cells in vivo.

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Cytomegalovirus Replication in Semen Is Associated with Higher Levels of Proviral HIV DNA and CD4+T Cell Activation during Antiretroviral Treatment

Journal of Virology ◽

10.1128/jvi.00831-14 ◽

2014 ◽

Vol 88 (14) ◽

pp. 7818-7827 ◽

Cited By ~ 46

Author(s):

Sara Gianella ◽

Marta Massanella ◽

Douglas D. Richman ◽

Susan J. Little ◽

Celsa A. Spina ◽

...

Keyword(s):

T Cells ◽

T Cell Activation ◽

Immune Activation ◽

Genital Tract ◽

Cell Activation ◽

Future Studies ◽

Hiv Rna ◽

Hiv Dna ◽

Dna Reservoir

ABSTRACTAsymptomatic cytomegalovirus (CMV) replication occurs frequently in the genital tract in untreated HIV-infected men and is associated with increased immune activation and HIV disease progression. To determine the connections between CMV-associated immune activation and the size of the viral reservoir, we evaluated the interactions between (i) asymptomatic seminal CMV replication, (ii) levels of T cell activation and proliferation in blood, and (iii) the size and transcriptional activity of the HIV DNA reservoir in blood from 53 HIV-infected men on long-term antiretroviral therapy (ART) with suppressed HIV RNA in blood plasma. We found that asymptomatic CMV shedding in semen was associated with significantly higher levels of proliferating and activated CD4+T cells in blood (P< 0.01). Subjects with detectable CMV in semen had approximately five times higher average levels of HIV DNA in blood CD4+T cells than subjects with no CMV. There was also a trend for CMV shedders to have increased cellular (multiply spliced) HIV RNA transcription (P= 0.068) compared to participants without CMV, but it is unclear if this transcription pattern is associated with residual HIV replication. In multivariate analysis, the presence of seminal plasma CMV (P= 0.04), detectable 2-long terminal repeat (2-LTR), and lower nadir CD4+(P< 0.01) were independent predictors of higher levels of proviral HIV DNA in blood. Interventions aimed at reducing seminal CMV and associated immune activation may be important for HIV curative strategies. Future studies of anti-CMV therapeutics will help to establish causality and determine the mechanisms underlying these described associations.IMPORTANCEAlmost all individuals infected with HIV are also infected with cytomegalovirus (CMV), and the replication dynamics of the two viruses likely influence each other. This study investigated interactions between asymptomatic CMV replication within the male genital tract, levels of inflammation in blood, and the size of the HIV DNA reservoir in 53 HIV-infected men on long-term antiretroviral therapy (ART) with suppressed HIV RNA in blood plasma. In support of our primary hypothesis, shedding of CMV DNA in semen was associated with increased activation and proliferation of T cells in blood and also significantly higher levels of HIV DNA in blood cells. These results suggest that CMV reactivation might play a role in the maintenance of the HIV DNA reservoir during suppressive ART and that it could be a target of pharmacologic intervention in future studies.

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Allogeneic Hematopoietic Stem Cell Transplantation Cures CLL: A Retrospective Analysis from the SFGM-TC Registry.

Blood ◽

10.1182/blood.v108.11.2991.2991 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2991-2991

Author(s):

Mauricette Michallet ◽

Quoc-Hung Lê ◽

Jean-Paul Vernant ◽

Franck E. Nicolini ◽

Jean-Luc Harousseau ◽

...

Keyword(s):

Autologous Transplantation ◽

Disease Status ◽

Allogeneic Transplantation ◽

Cell Transplant ◽

Hematopoietic Stem ◽

Cord Blood Cell ◽

The Status ◽

The Impact ◽

Long Term Survivors

Abstract This retrospective study concerned 471 B-CLL patients registered in the SFGM-TC registry from Apr 1,984 to Feb 2,005, who underwent either autologous transplantation (n=313, 138 F and 175 M, median age = 54, 236 PBSC and 77 BM) or allogeneic transplantation (n=158, 78 F and 80 M, median age = 49, 76 PBSC, 81 BM and 1 cord blood cell transplant from 17 related and 141 unrelated donors). Among alloT patients, 50 were ABO incompatible and 70 sex-mismatched. The median interval diagnosis-transplantation was 32 months for autoT and 51 months for alloT. Just before conditioning 302 autoT and 143 alloT were evaluated for the disease status: 100 and 26 patients were in CR, 170 and 55 were in PR, 4 and 13 in stable disease (SD), 28 and 49 in progressive disease (PD) for autoT and alloT respectively. Among alloT patients, 73 received reduced intensity conditioning (RIC) and 85 standard conditioning (72 Cyt+TBI, 33 Fluda+TBI, 23 Fluda+Bu+ATG, 8 Cyt+Bu and 21 other). Before autoT the conditioning consisted of 224 Cyt+TBI, 45 BEAM and 44 other. After alloT, 71 patients developed an aGVHD ≥ grade II and 60 developed a cGVHD (25 limited and 35 extensive). The non-relapse mortality at 1 year was 29%. With a mean follow-up of 28 months for autoT and 40 months for alloT, the probabilities of 3-year, 5-year and 8-year overall survival were 80%, 66%, 45.5% after autoT and 52%, 48% and 35% after alloT respectively. An analysis aimed to determine the percentage of long-term survivors, or patients censored on the final plateau of survival curves was performed on alloT and autoT groups. A mixture model, gfcure with Splus statistical package determined the percentages of long-term survivors and its adequacy was verified graphically. The percentage of long-term survivors for the autoT group was 1.2%, with a mean survival length for uncured population of 160 months. Fig A shows that both curves were close and consequently shows good adequacy and the absence of a final plateau. The percentage of long-term survivors for alloT was 34.03% (figure1). Fig B shows rather good adequacy. The study of the impact of usual prognosis factors (age, time diagnosis-transplant, sex match, HLA match, CMV status, type of conditioning, BM or PBSC, ABO compatibility and disease status before transplantation) on the percentage of long-term survivors showed that only the status of disease at transplant had a significant impact: (CR vs SD or PD, HR: 0.11 [0.02–0.5] p=0.01 and PR vs SD or PD, HR: 0.30 [0.09–0.96] p=0.04). This study pointed out the possibility of curing B-CLL patients who responded to conventional chemotherapy with allogeneic transplantation rather than with autologous transplantation. Figure Figure Figure Figure

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A Modified HIV1-Based Lentiviral Vector Can Transduce Rhesus Hematopoietic Repopulating Cells as Efficiently as An SIV Vector in An Autologous Transplantation Model.

Blood ◽

10.1182/blood.v114.22.692.692 ◽

2009 ◽

Vol 114 (22) ◽

pp. 692-692

Author(s):

Naoya Uchida ◽

Phillip W Hargrove ◽

Kareem Washington ◽

Coen J. Lap ◽

Matthew M. Hsieh ◽

...

Keyword(s):

T Cells ◽

Blood Cells ◽

Conflicts Of Interest ◽

Lentiviral Vector ◽

Autologous Transplantation ◽

Vector System ◽

Transduction Efficiency ◽

Large Animal ◽

Hematopoietic Stem ◽

Cd34 Cells

Abstract Abstract 692 HIV1-based vectors transduce rhesus hematopoietic stem cells poorly due to a species specific block by restriction factors, such as TRIM5αa which target HIV1 capsid proteins. The use of simian immunodeficiency virus (SIV)-based vectors can circumvent this restriction, yet use of this system precludes the ability to directly evaluate HIV1-based lentiviral vectors prior to their use in human clinical trials. To address this issue, we previously developed a chimeric HIV1 vector (χHIV vector) system wherein the HIV1-based lentiviral vector genome is packaged in the context of SIV capsid sequences. We found that this allowed χHIV vector particles to escape the intracellular defense mechanisms operative in rhesus hematopoietic cells as judged by the efficient transduction of both rhesus and human CD34+ cells. Following transplantation of rhesus animals with autologous cell transduced with the χHIV vector, high levels of marking were observed in peripheral blood cells (J Virol. 2009 Jul. in press). To evaluate whether χHIV vectors could transduce rhesus blood cells as efficiently as SIV vectors, we performed a competitive repopulation assay in two rhesus macaques for which half of the CD34+ cells were transduced with the standard SIV vector and the other half with the χHIV vector both at a MOI=50 and under identical transduction conditions. The transduction efficiency for rhesus CD34+ cells before transplantation with the χHIV vector showed lower transduction rates in vitro compared to those of the SIV vector (first rhesus: 41.9±0.83% vs. 71.2±0.46%, p<0.01, second rhesus: 65.0±0.51% vs. 77.0±0.18%, p<0.01, respectively). Following transplantation and reconstitution, however, the χHIV vector showed modestly higher gene marking levels in granulocytes (first rhesus: 12.4% vs. 6.1%, second rhesus: 36.1% vs. 27.2%) and equivalent marking levels in lymphocytes, red blood cells (RBC), and platelets, compared to the SIV vector at one month (Figure). Three to four months after transplantation in the first animal, in vivo marking levels plateaued, and the χHIV achieved 2-3 fold higher marking levels when compared to the SIV vector, in granulocytes (6.9% vs. 2.8%) and RBCs (3.3% vs. 0.9%), and equivalent marking levels in lymphocytes (7.1% vs. 5.1%) and platelets (2.8% vs. 2.5)(Figure). Using cell type specific surface marker analysis, the χHIV vector showed 2-7 fold higher marking levels in CD33+ cells (granulocytes: 5.4% vs. 2.7%), CD56+ cells (NK cells: 6.5% vs. 3.2%), CD71+ cells (reticulocyte: 4.5% vs. 0.6%), and RBC+ cells (3.6% vs. 0.9%), and equivalent marking levels in CD3+ cells (T cells: 4.4% vs. 3.3%), CD4+ cells (T cells: 3.9% vs. 4.6%), CD8+ cells (T cells: 4.2% vs. 3.9%), CD20+ cells (B cells: 7.6% vs. 4.8%), and CD41a+ cells (platelets: 3.5% vs. 2.2%) 4 months after transplantation. The second animal showed a similar pattern with higher overall levels (granulocytes: 32.8% vs. 19.1%, lymphocytes: 24.4% vs. 17.6%, RBCs 13.1% vs. 6.8%, and platelets: 14.8% vs. 16.9%) 2 months after transplantation. These data demonstrate that our χHIV vector can efficiently transduce rhesus long-term progenitors at levels comparable to SIV-based vectors. This χHIV vector system should allow preclinical testing of HIV1-based therapeutic vectors in the large animal model, especially for granulocytic or RBC diseases. Disclosures: No relevant conflicts of interest to declare.

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RNA-seq of Human T-Cells After Hematopoietic Stem Cell Transplantation Identifies Linc00402 as a Novel Regulator of T-Cell Alloimmunity

10.1101/2020.04.16.045567 ◽

2020 ◽

Author(s):

Daniel Peltier ◽

Molly Radosevich ◽

Guoqing Hou ◽

Cynthia Zajac ◽

Katherine Oravecz-Wilson ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Solid Organ Transplantation ◽

Solid Organ ◽

Hematopoietic Stem ◽

Cell Responses ◽

Human T Cells ◽

Allogeneic Stimulation

ABSTRACTMechanisms governing allogeneic T-cell responses after allogeneic hematopoietic stem cell (HSC) and solid organ transplantation are incompletely understood. Long non-coding RNAs (lncRNA) do not code for, but control gene expression with tissue specificity. However, their role in T-cell alloimmunity is unknown. We performed RNA-seq on donor T-cells from HSCT patients and found that increasing strength of allogeneic stimulation caused greater differential expression of lncRNAs. The differential expression was validated in an independent patient cohort, and also following ex vivo allogeneic stimulation of healthy human T-cells. Linc00402, a novel, conserved lncRNA, was identified as the most differentially expressed and was enriched 88 fold in human T-cells. Mechanistically, it was mainly located in the cytoplasm, and its expression was rapidly reduced following T-cell activation. Consistent with this, tacrolimus preserved the expression of Linc00402 following T-cell activation, and lower levels of Linc00402 were found in patients who subsequently went on to develop acute graft versus host disease (GVHD). The dysregulated expression of Linc00402 was also validated in murine T-cells, both in vitro and in vivo. Functional studies using multiple modalities to deplete Linc00402 in both mouse and human T-cells, demonstrated a critical role for Linc00402 in the T-cell proliferative response to an allogeneic stimulus but not a non-specific anti-CD3/CD28 stimulus. Thus, our studies identified Linc00402 as a novel, conserved regulator of allogeneic T-cell function. Because of its T-cell specific expression and its impact on allogeneic T-cell responses, targeting Linc00402 may improve outcomes after allogeneic HSC and solid organ transplantation.One sentence summaryLncRNAs are differentially expressed by allogeneic antigen-stimulated T-cells, and the novel lncRNA, Linc00402, is a specific regulator of mouse and human allogeneic T-cells.

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Autologous transplantation of canine long-term marrow culture cells genetically marked by retroviral vectors

Blood ◽

10.1182/blood.v79.2.356.bloodjournal792356 ◽

1992 ◽

Vol 79 (2) ◽

pp. 356-364 ◽

Cited By ~ 1

Author(s):

RF Carter ◽

AC Abrams-Ogg ◽

JE Dick ◽

SA Kruth ◽

VE Valli ◽

...

Keyword(s):

Gene Transfer ◽

Autologous Transplantation ◽

Large Animal Model ◽

Multiple Infections ◽

Large Animal ◽

Retroviral Infection ◽

Hematopoietic Stem ◽

Marrow Cells

Retroviral infection of bone marrow cells in long-term marrow cultures (LTMCs) offers several theoretical advantages over other methods for gene transfer into hematopoietic stem cells. To investigate the feasibility of this approach in a large animal model system, we subjected LTMCs from nine dogs to multiple infections with retrovirus containing the neomycin phosphotransferase gene (neo) during 21 days of culture. Feeder layers, cocultivation, polycations, and selection were not used. The in vitro gene transfer efficiency was 70% as determined by polymerase chain reaction amplification of neo sequences in colony- forming unit granulocyte-macrophage (CFU-GM) obtained from day-21 LTMCs. Day-21 LTMC cells were infused into autologous recipients with (four dogs) and without (three dogs) marrow-ablative conditioning. At 3 months posttransplant, up to 10% of marrow cells contained the neo gene. This percentage declined to 0.1% to 1% at 10 to 21 months posttransplant. Neo was also detected in individual CFU-GM, burst- forming unit-erythroid (BFU-E), and CFU-Mix progenitors derived from marrow up to 21 months postinfusion and in cultures of peripheral blood- derived T cells up to 19 months postinfusion. There was no difference in the percentage of neo-marked cells present when dogs that received marrow ablative conditioning were compared with dogs receiving no conditioning. Detection of neo-marked marrow cells almost 2 years after autologous transplantation in a large mammalian species shows that retroviral infection of marrow cells in LTMCs is a potentially nontoxic and efficient protocol for gene transfer. Further, our results suggest that marrow conditioning and in vivo selection pressure to retain transplanted cells may not be absolute requirements for the retention of genetically marked cells in vivo.

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The Innate Immune Sensor Sting Promotes Donor CD8+ T Cell Activation and Recipient APC Death Early after Preclinical Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽

10.1182/blood-2019-130197 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3202-3202

Author(s):

Cameron S. Bader ◽

Henry Barreras ◽

Casey O. Lightbourn ◽

Sabrina N. Copsel ◽

Dietlinde Wolf ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

T Cell Activation ◽

Cd8 T Cells ◽

Research Funding ◽

Cell Activation ◽

Cd8 T Cell ◽

Advisory Committees ◽

Hematopoietic Stem

Graft-versus-host disease (GVHD) remains a significant cause of morbidity and mortality in patients receiving allogeneic hematopoietic stem cell transplants (aHSCTs). Pre-HSCT conditioning typically consists of irradiation and drug administration resulting in the death of rapidly dividing cells and release of endogenous danger signals. These molecules drive the activation of antigen presenting cells (APCs) and the differentiation of allo-reactive donor T cells, leading to damage of particular host tissues characteristic of GVHD. Cell death following conditioning has promoted the hypothesis that sensors of cytoplasmic DNA damage in GVHD target tissues contribute to pro-inflammatory cytokine production. We identified a role for Stimulator of Interferon Genes (STING), an innate immune sensor, in GVHD using pre-clinical MHC-matched unrelated donor (MUD) aHSCT models. Here we show that STING rapidly promotes donor CD8+ T cell activation and recipient APC death early after aHSCT. To assess STING involvement immediately post-HSCT, cytokine mRNA expression was examined 48 hrs after transplant of MUD C3H.SW bone marrow (BM) + T cells into irradiated B6 wildtype (WT) or STING-/- recipients. Colon tissue from STING-/- recipients had >2x reduction in IFNβ, TNFα and IL-6 mRNA vs WT. MUD STING-/- HSCT recipients also experienced decreased weight loss, GVHD scores and skin pathology 6 wks post-HSCT vs WT. Double chimerism studies showed that the absence of STING in non-hematopoietic cells was responsible for GVHD amelioration. Conversely, a single dose of the highly specific STING agonist DMXAA given in vivo increased IFNβ, TNFα and IL-6 mRNA expression in WT, but not STING-/-, colon tissue 48 hrs after transplant and increased GVHD scores and lethality post-HSCT. Post-transplant cytoxan treatment abolished the ability of DMXAA to augment GVHD, supporting the notion that STING signaling increases donor T cell activation during aHSCT. To evaluate the potential impact of STING in the clinical setting, we transplanted C3H.SW BM + T cells into mice homozygous for a murine homologue of a human allele associated with diminished STING activity (STINGHAQ/HAQ) and found that these mice also exhibited diminished GVHD. Interestingly, our findings that STING deficiency ameliorates GVHD in MUD aHSCT contrasts to reported observations that STING deficiency can exacerbate GVHD after MHC-mismatched (MMUD) aHSCT (Fischer J, et al, Sci. Transl. Med. 2017). Since CD4+ and CD8+ T cells are central in MMUD and MUD GVHD, respectively, we hypothesized that STING's effect on the predominant T cell subset in each model may explain these seemingly paradoxical results in STING-/- vs WT recipients. Therefore, we transplanted MMUD BALB/c BM + CD8+ T cells into B6-WT and STING-/- mice and found that - in contrast to MMUD recipients of combined CD4+ and CD8+ T cells - STING-/- recipients developed lower GVHD clinical scores, reduced skin pathology and had lower frequencies of activated T cells 8 wks post-HSCT vs WT, supporting a role for STING in the promotion of CD8+ T cell-mediated GVHD. Next, we investigated if recipient APCs played a role in STING's enhancement of CD8+ T cell-mediatedGVHD. We found that STING-/- mice had greater frequencies and numbers of recipient splenic CD11b+CD11c+ APCs 1 day after MMUD B6 into BALB/c aHSCT (Fig. A). BALB/c-STING-/- APCs also expressed reduced MHC class I protein levels (Fig. B). Moreover, STING-/- recipient spleens contained lower numbers of donor CD8+ T cells producing IFNγ and TNFα (Fig. C). These data support the hypothesis that STING contributes to early activation of donor CD8+ T cells and elimination of recipient APCs. Next, to identify if the loss of host MHC II+ APCs affected subsequent donor CD4+ T cell activation, B6-Nur77GFP transgenic donor T cells were used to explicitly monitor T cell receptor signaling. Consistent with increased numbers of host MHC II+ APCs in the spleens of STING-/- recipients 1 day post-aHSCT, we found greater frequencies and numbers of donor Nur77GFP CD4+ T cells expressing GFP, CD69 and IFNγ in STING-/- spleens 6 days after transplant (Fig. D). In summary, our studies demonstrate that STING plays an important role in regulating aHSCT and provide one potential mechanism by which STING could promote CD8+ T cell-mediated GVHD yet diminish CD4+-mediated GVHD. Overall, our studies suggest this pathway can provide a target for new therapeutic strategies to ameliorate GVHD. Disclosures Blazar: BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees; Childrens' Cancer Research Fund: Research Funding; KidsFirst Fund: Research Funding; Tmunity: Other: Co-Founder; Kamon Pharmaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees; Regeneron Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Five Prime Therapeutics Inc: Co-Founder, Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics and BlueRock Therapeuetics: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc.: Research Funding; RXi Pharmaceuticals: Research Funding; Alpine Immune Sciences, Inc.: Research Funding; Abbvie Inc: Research Funding; Leukemia and Lymphoma Society: Research Funding. Levy:Heat Biologics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pelican Therapeutics: Consultancy, Research Funding.

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